Skin Mast Cells Contribute to Sporothrix schenckii Infection

Background: Sporothrix schenckii (S. schenckii), a dimorphic fungus, causes sporotrichosis. Mast cells (MCs) have been described to be involved in skin fungal infections. The role of MCs in cutaneous sporotrichosis remains largely unknown. Objectives: To characterize the role and relevance of MCs in cutaneous sporotrichosis. Methods: We analyzed cutaneous sporotrichosis in wild-type (WT) mice and two different MC-deficient strains. In vitro, MCs were assessed for S. schenckii-induced cytokine production and degranulation after incubation with S. schenckii. We also explored the role of MCs in human cutaneous sporotrichosis. Results: WT mice developed markedly larger skin lesions than MC-deficient mice (> 1.5 fold) after infection with S. schenckii, with significantly increased fungal burden. S. schenckii induced the release of tumor necrosis factor alpha (TNF), interleukin (IL)-6, IL-10, and IL-1β by MCs, but not degranulation. S. schenckii induced larger skin lesions and higher release of IL-6 and TNF by MCs as compared to the less virulent S. albicans. In patients with sporotrichosis, TNF and IL-6 were increased in skin lesions, and markedly elevated levels in the serum were linked to disease activity. Conclusions: These findings suggest that cutaneous MCs contribute to skin sporotrichosis by releasing cytokines such as TNF and IL-6

Bruton's tyrosine kinase inhibition—An emerging therapeutic strategy in immune-mediated dermatological conditions

Funding Information: PM‐B received honoraria for acting as a consultant and/or as a speaker for AbbVie, Janssen, Novartis, LEO Pharma, Almirall, Sanofi, Viatris, L’Oréal, and Cantabria Labs and served as a principal investigator in clinical trials supported by AbbVie, Sanofi, and Novartis. AB received honoraria for lectures and educational events for LEO Pharma, Janssen‐Cilag, AbbVie, and Novartis. PK received payment/honoraria for lectures/presentations outside of submitted work for Novartis and Roche.. SM‐R received funding from GA²LEN Global Allergy and Asthma European Network. MM served as a speaker and/or advisor for and/or has received research funding from Allakos, Amgen, Aralez, ArgenX, AstraZeneca, Blueprint, Celldex, Centogene, CSL Behring, FAES, Genentech, Gilead, GIInnovation, Innate Pharma, Kyowa Kirin, Leo Pharma, Lilly, Menarini, Moxie, Novartis, Roche, Sanofi/Regeneron, Third Harmonic Bio, UCB, and Uriach. SF and JS have no conflicts of interest to disclose. Funding Information: We acknowledge Becky Fox‐Spencer, Ph.D. (on behalf of Bedrock Healthcare Communications), who provided writing support for this manuscript, funded by Novartis Pharma AG (Basel, Switzerland). Publisher Copyright: © 2022 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.Bruton's tyrosine kinase (BTK), a member of the Tec kinase family, is critically involved in a range of immunological pathways. The clinical application of BTK inhibitors for B-cell malignancies has proven successful, and there is strong rationale for the potential benefits of BTK inhibitors in some autoimmune and allergic conditions, including immune-mediated dermatological diseases. However, the established risk-to-benefit profile of “first-generation” BTK inhibitors cannot be extrapolated to these emerging, non-oncological, indications. “Next-generation” BTK inhibitors such as remibrutinib and fenebrutinib entered clinical development for chronic spontaneous urticaria (CSU); rilzabrutinib and tirabrutinib are being studied as potential treatments for pemphigus. Promising data from early-phase clinical trials in CSU suggest potential for these agents to achieve strong pathway inhibition, which may translate into measurable clinical benefits, as well as other effects such as the disruption of autoantibody production. BTK inhibitors may help to overcome some of the shortcomings of monoclonal antibody treatments for immune-mediated dermatological conditions such as CSU, pemphigus, and systemic lupus erythematosus. In addition, the use of BTK inhibitors may improve understanding of the pathophysiological roles of mast cells, basophils, and B cells in such conditions.publishersversionepub_ahead_of_prin

STAT3 gain-of-function is not responsible for low total IgE levels in patients with autoimmune chronic spontaneous urticaria

BackgroundThe pathogenesis of chronic spontaneous urticaria (CSU) has not been clarified entirely. Type IIb autoimmune chronic spontaneous urticaria (CSUaiTIIb) is a distinct subtype of CSU that is often difficult to treat and is connected to low levels of total IgE. Previous findings indicate that an enhanced signal transducer and activator of transcription 3 (STAT3) may be responsible for reduced IgE serum levels.ObjectiveOur aim was to investigate a possible underlying gain-of-function mutation or activating polymorphism in STAT3 that could be responsible for the low levels of IgE in patients with CSUaiTIIb.MethodsWe included 10 patients with CSUaiTIIb and low levels of IgE and sequenced selected single nucleotide polymorphisms (SNP) in STAT3 associated with common autoimmune diseases. Exon sequencing was performed for the most relevant exons of STAT3. To test for a gain-of-function of STAT3, we performed a phospho-specific flow cytometry analysis of STAT3 in peripheral blood mononuclear cells before and after stimulation with interleukin-6.ResultsNo differences were found in the prevalence of the tested SNPs between our patients and a control population. Moreover, we could not find any mutations or variants on the tested exons of STAT3. The function of STAT3 was also not altered in our patients.ConclusionIn total, we could not find any evidence for our hypothesis that low IgE in patients with CSUaiTIIb is linked to mutations in STAT3 or altered activity of STAT3. Thus, it remains to be discovered what causes the low serum levels of IgE in patients with CSUaiTIIb

Topical inflammasome inhibition with disulfiram prevents irritant contact dermatitis

Background: The pathogenesis of contact dermatitis, a common inflammatory skin disease with limited treatment options, is held to be driven by inflammasome activation induced by allergens and irritants. We here aim to identify inflammasome-targeting treatment strategies for irritant contact dermatitis. Methods: A high content screen with 41,184 small molecules was performed using fluorescent Apoptosis associated speck-like protein containing a CARD (ASC) speck formation as a readout for inflammasome activation. Hit compounds were validated for inhibition of interleukin (IL)-1β secretion. Of these, the approved thiuramdisulfide derivative disulfiram was selected and tested in a patch test model of irritant contact dermatitis in 25 healthy volunteers. Topical application of disulfiram, mometasone or vehicle was followed by application of sodiumdodecylsulfate (SDS) for 24 h each. Eczema induction was quantified by mexameter and laser speckle imaging. Corneocyte sampling of lesional skin was performed to assess inflammasome-mediated cytokines IL-1β and IL-18. Results: Disulfiram induced a dose-dependent inhibition of ASC speck formation and IL-1β release in cellular assays in vitro. In vivo, treatment with disulfiram, but not with vehicle and less mometasone, inhibited SDS-induced eczema. This was demonstrated by significantly lower erythema and total perfusion values assessed by mexameter and laser speckle imaging for disulfiram compared to vehicle (p < 0.001) and/or mometasone (p < 0.001). Also, corneocyte IL-18 levels were significantly reduced after application of disulfiram compared to vehicle (p < 0.001). Conclusion: We show that disulfiram is a dose-dependent inhibitor of inflammasome pathway activation in vitro and inhibitor of SDS-induced eczema in vivo. Topical application of disulfiram represents a potential treatment option for irritant contact dermatitis

The Number of MRGPRX2-Expressing Cells Is Increased in Skin Lesions of Patients With Indolent Systemic Mastocytosis, But Is Not Linked to Symptom Severity

BackgroundRecently, the expression of the mast cell (MC) receptor Mas-related G protein–coupled receptor X2 (MRGPRX2) has been detected in lesional skin of adult patients with cutaneous mastocytosis. As of yet, little is known about the clinical relevance of MRGPRX2 and its agonists in patients with mastocytosis, including indolent systemic mastocytosis (ISM).MethodsMRGPRX2 and MRGPRX2 agonists, cortistatin (CST), and major basic protein (MBP) were analyzed in lesional and non-lesional skin of patients with ISM and skin of healthy controls by immunohistochemistry. Co-localization of MRGPRX2 and MRGPRX2-mRNA with the MC marker tryptase was assessed by immunofluorescence microscopy and in situ hybridization, respectively. We assessed clinical, demographic, and laboratory data, including mastocytosis activity score (MAS), serum tryptase, and KIT D816V allele burden.ResultsThe number of MRGPRX2-expressing (MRGPRX2+) cells, MRGPRX2-mRNA+ MCs, and CST-expressing (CST+) and MBP-expressing (MBP+) cells was significantly higher in lesional skin as compared to non-lesional skin and/or skin of healthy controls (all p &lt; 0.05). Increased numbers of MRGPRX2+ cells, MRGPRX2-mRNA+ MCs, and CST+ and MBP+ cells were not associated with clinical and laboratory features of ISM, including disease burden, symptom severity, evidence of anaphylaxis, and tryptase levels.ConclusionsSkin lesions of patients with ISM showed high numbers of MRGPRX2+ cells, although they were not linked to symptom severity. Clinical relevance of the MRGPRX2-mediated pathway of MC activation in ISM remains unclear and should be investigated in further studies

SARS-CoV-2 in severe COVID-19 induces a TGF-β-dominated chronic immune response that does not target itself

The pathogenesis of severe COVID-19 reflects an inefficient immune reaction to SARS-CoV-2. Here we analyze, at the single cell level, plasmablasts egressed into the blood to study the dynamics of adaptive immune response in COVID-19 patients requiring intensive care. Before seroconversion in response to SARS-CoV-2 spike protein, peripheral plasmablasts display a type 1 interferon-induced gene expression signature; however, following seroconversion, plasmablasts lose this signature, express instead gene signatures induced by IL-21 and TGF-β, and produce mostly IgG1 and IgA1. In the sustained immune reaction from COVID-19 patients, plasmablasts shift to the expression of IgA2, thereby reflecting an instruction by TGF-β. Despite their continued presence in the blood, plasmablasts are not found in the lungs of deceased COVID-19 patients, nor does patient IgA2 binds to the dominant antigens of SARS-CoV-2. Our results thus suggest that, in severe COVID-19, SARS-CoV-2 triggers a chronic immune reaction that is instructed by TGF-β, and is distracted from itself

Dephosphorylation of the adapter protein Bcl-10 by the Ser/Thr-Phosphatase calcineurin is essential for NF-kB activation in Th-lymphocytes

Durch Aufklärung von Signalübertragungsprozessen wird es möglich, den detaillierten molekularen Ablauf von Immunreaktionen zu rekonstruieren. Zudem bekommt man Aufschluss über Fehlfunktionen des Immunsystems was dabei hilft, therapeutische Ansätze zur Behandlung von Immunerkrankungen zu entwickeln. Für die Identifizierung und pharmakologische Intervention von gestörten Signalwegsprozessen werden häufig niedermolekulare, zellpermeable Inhibitoren benutzt. Deren Signalwegsspezifität ist ein wichtiges Kriterium für ihre Anwendung in der Forschung und Klinik. In dieser Arbeit wurden zum einen Inhibitoren des Calcineurin (CaN)/NFAT-Signalwegs hinsichtlich ihrer Kreuzreaktivität mit der T-Zell-Rezeptor-vermittelten Aktivierung der NF-κB und AP-1 Signalübertragungswege untersucht. Zum anderen wurde analysiert, an welcher Stelle des NF-κB Signalweges Calcineurin aktivierend wirkt. Für die Analyse der Kreuzreaktivität wurden die Inhibitoren AM404, BTP1, CsA, NCI3 und INCA6 untersucht. Durch Etablierung und Verwendung der „Fluorescent Cell Barcoding“-Technik konnte die Aktivierung bzw. Hemmung von NF-κB p65, p38 und ERK1/2 gleichzeitig auf Einzelzellebene bestimmt werden. Die Analysen zeigten, dass sich AM404, BTP1 und NCI3 für eine spezifische CaN/NFAT-Inhibierung eignen, während INCA6 und CsA auch die NF-κB-und AP-1-Aktivierung blockieren. Mit Hilfe des Calcineurin Inhibitors CsA konnte gezeigt werden, dass diese Ser/Thr Phosphatase an der Position des Adapterproteins Bcl-10 aktivierend auf den NF-κB Signalweg wirkt. Eine CsA Behandlung der Zellen beeinträchtigt daher die nachfolgenden Signalwegsprozesse wie IKKβ-Phosphorylierung, IκBα- Degradation, sowie Kerntranslokation und DNA-Bindung von NF-κB p65. Durch Ko- Immunopräzipitation konnte eine konstitutive Assoziation von CaN mit dem Bcl-10/MALT1-Komplex nachgewiesen werden. Diese Interaktion ist eine Voraussetzung für die Dephosphorylierung von phospho-Bcl-10 am Ser138, die bereits als eine entscheidende post-translationale Bcl-10-Modifikation für die Aktivierung von NF-κB identifiziert wurde. Die bessere Kenntnis des molekularen Mechanismus der TZR-gesteuerten NF-κB Aktivierung ermöglicht die Entwicklung neuer therapeutischer Ansätze, deren Target CaN und/oder Bcl-10 ist, zur gezielten Inhibierung von NF-κB-Signalprozessen bei gestörten Immunreaktionen oder Lymphomen.The analysis of signaling processes allows the detailed reconstruction of molecular sequences of immune reactions. Thereby, it is possible to gain insight into immune-dysfunctions which will help to develop new therapeutic strategies for the treatment of autoimmune diseases. Low molecular and cell permeable inhibitors are widely used for identification and pharmacological intervention of signaling processes. Therefore, pathway specificity of inhibitors is important for clinical as well as for research applications. Here, inhibitors of the calcineurin (CaN)/NFAT-signaling pathway were analyzed with regard to their cross reactivity on T-cell-receptor mediated activation of the NF-κB and AP-1 signaling pathways. Further, it was studied at which position in the NF-κB pathway CaN exerts it´s activating function. For the analysis of cross reactivity, the inhibitors AM404, BTP1, CsA, NCI3, and INCA6 were used. Establishment of the Fluorescent cell barcoding technique allowed the simultaneous single cell measurement of activation or inhibition levels of NF-κB p65, p38, and pERK1/2. The analyses show that the inhibitors AM404 and BTP1 and NCI3 are suitable for specific inhibition of CaN/NFAT, whereas INCA6 and CsA also block the activation of NF-κB and AP-1. Using the CaN inhibitor CsA it could be shown, that the adapter protein Bcl-10 is the pathway position where this Ser/Thr phosphatase contributes to the activation of the NF-κB signaling pathway. Consequently, CsA treatment impaired downstream signaling processes like IKKβ-phosphorylation, IκBα-degradation, as well as nuclear translocation and NF-κB p65 DNA binding. By co-immunoprecipitation a constitutive association of CaN with the Bcl-10/MALT1-complex was shown. This association is a prerequisite for dephosphorylation of phosphorylated Bcl-10 Ser138, which was already identified as an important post translational modification essential for NF-κB activation. The better knowledge of the molecular processes of TCR-mediated NF-κB-activation allows the development of new therapeutic approaches which target CaN and/or Bcl-10 for the specific inhibition of NF-κB signaling processes during immune-dysfunctions or lymphomas